Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization.

RNA polymerase II (Pol II) transcribes eukaryotic mRNA genes. To initiate transcription, pre-initiation complexes (PICs) containing Pol II and general transcription factors (GTFs) form on the core promoters of target genes. In cells this process is regulated by transcriptional activators, co-activators, and chromatin modifying complexes. Reconstituted in vitro transcription systems are important tools for studying the enzymology and fundamental steps in the transcription reaction. In these systems, studying transcription can be complex due to the heterogeneous mixture of transcriptionally active and inactive complexes that assemble at promoters. Accordingly, we developed a technique to use single molecule microscopy to resolve this heterogeneity and distinguish transcriptionally active complexes from inactive complexes. This system uses fluorescently-labeled promoter DNA and a minimal reconstituted transcription system consisting of purified human Pol II and GTFs. Here we describe the materials, methods, and analysis required to study Pol II transcription at the single molecule level. The flexibility of our single molecule method allows for adaptation to answer diverse mechanistic questions about transcription that would otherwise be difficult to study using ensemble assays.